Abstract Fatty acids (FAs) play a key role in cancer metabolism since they are essential for signal transduction, energy production, and membrane biogenesis. In general, cells acquire FAs from exogenous sources via CD36- mediated uptake and/or endogenously through de novo lipogenesis involving FA synthase (FASN). High levels of FASN expression are associated with poor prognosis in various cancers, including breast cancer. In turn, FASN inhibition can induce apoptosis in cancer cells. While the functional significance of FASN in cancer metabolism has been extensively studied, the role of CD36 in cancer progression remains poorly understood. Through studies in HER2+ breast cancer cells, we have found that during acquisition of resistance to the HER2 inhibitor lapatinib, breast cancer cells may activate the CD36-mediated pathway to obtain FAs independently of de novo synthesis. HER2 is a receptor tyrosine kinase overexpressed in 25% of breast cancers. HER2 directly phosphorylates and activates FASN, while also promoting FASN gene expression. In our preliminary studies, we found that 1) upon acquiring resistance to lapatinib, cells are also refractory to FASN inhibition, 2) expression of CD36 is significantly enhanced in lapatinib-resistant cells as well as in lapatinib-resistant mouse tumors, and 3) siRNA-mediated CD36 knockdown induces robust apoptosis in lapatinib-resistant cells, but not in sensitive cells. We hypothesize that there is a metabolic shift in lapatinib-resistant cells toward reliance on CD36-mediated FA uptake over de novo FA synthesis for maintaining the cellular FA pool ? consequently, CD36 is required for HER2+ breast tumor cells to develop resistance to HER2 inhibitors. The overarching goal of the current proposal is to establish an in vivo role of CD36 in resistance to lapatinib using two mouse models of HER2+ breast tumors: a) mammary gland specific CD36 knockout mice in a HER2/neu mammary tumor model (MMTV-neu/CD36KO) and b) a tumor xenograft model with CD36KO breast cancer cell lines created by CRISPR/Cas9 technology. To separate the FA transporter function of CD36 from its other functions, we will employ isocaloric low- and high- fat diets. Using these novel tumor models, we will determine the impact of CD36 loss in HER2+ breast tumor development (Aim 1) and the role of CD36 in breast tumor resistance to HER2 inhibitors (Aim 2). Given results from our cell culture models, the proposed research will be the logical next step toward understanding an in vivo function of CD36 in HER2+ breast tumor development and resistance to HER2 inhibitors. If our proposed studies validate the significance of CD36 in mediating acquired HER2 inhibitor resistance in vivo, it will also provide a rationale for developing a novel therapeutic CD36 inhibitor for cancer treatment, a pharmacological option which is currently not available.